Hypoxia-induced P4HA1 overexpression promotes post-ischemic angiogenesis by enhancing endothelial glycolysis through downregulating FBP1.

BACKGROUND: Angiogenesis is essential for tissue repair in ischemic diseases, relying on glycolysis as its primary energy source. Prolyl 4-hydroxylase subunit alpha 1 (P4HA1), the catalytic subunit of collagen prolyl 4-hydroxylase, is a glycolysis-related gene in cancers. However, its role in glycolysis-induced angiogenesis remains unclear. METHODS: P4HA1 expression was modulated using adenoviruses. Endothelial angiogenesis was evaluated through 5-ethynyl-2'-deoxyuridine incorporation, transwell migration, and tube formation assays in vitro. In vivo experiments measured blood flow and capillary density in the hindlimb ischemia (HLI) model. Glycolytic stress assays, glucose uptake, lactate production, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) were employed to assess glycolytic capacity. Transcriptome sequencing, validated by western blotting and RT-PCR, was utilized to determine underlying mechanisms. RESULTS: P4HA1 was upregulated in endothelial cells under hypoxia and in the HLI model. P4HA1 overexpression promoted angiogenesis in vitro and in vivo, while its knockdown had the opposite effect. P4HA1 overexpression reduced cellular α-ketoglutarate (α-KG) levels by consuming α-KG during collagen hydroxylation. Downregulation of α-KG reduced the protein level of a DNA dioxygenase, ten-eleven translocation 2 (TET2), and its recruitment to the fructose-1,6-biphosphatase (FBP1) promoter, resulting in decreased FBP1 expression. The decrease in FBP1 enhanced glycolytic metabolism, thereby promoting endothelial angiogenesis. CONCLUSIONS: Hypoxia-induced endothelial P4HA1 overexpression enhanced angiogenesis by promoting glycolytic metabolism reprogramming through the P4HA1/α-KG/TET2/FBP1 pathway. The study's findings underscore the significance of P4HA1 in post-ischemic angiogenesis, suggesting its therapeutic potential for post-ischemic tissue repair.

TMT quantitative proteomics reveals key proteins relevant to microRNA-1-mediated regulation in osteoarthritis.

Osteoarthritis (OA) is the second-commonest arthritis, but pathogenic and regulatory mechanisms underlying OA remain incompletely understood. Here, we aimed to identify the mechanisms associated with microRNA-1 (miR-1) treatment of OA in rodent OA models using a proteomic approach. First, N = 18 Sprague Dawley (SD) rats underwent sham surgery (n = 6) or ACL transection (n = 12), followed at an interval of one week by randomization of the ACL transection group to intra-articular administration of either 50 µL placebo (control group) or miR-1 agomir, a mimic of endogenous miR-1 (experimental group). After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and immunohistochemically stained for the presence of MMP-13. Second, N = 30 Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 transgenic mice were randomized to intra-articular administration of either placebo (control group, N = 15) or tamoxifen, an inducer of miR-1 expression (experimental group, N = 15), before undergoing surgical disruption of the medial meniscus (DMM) after an interval of five days. After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and underwent differential proteomic analysis. Specifically, tandem mass tagging (TMT) quantitative proteomic analysis was employed to identify inter-group differentially-expressed proteins (DEP), and selected DEPs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) technology. Immunohistochemically-detected MMP-13 expression was significantly lower in the experimental rat group, and proteomic analyses of mouse tissue homogenate demonstrated that of 3526 identified proteins, 345 were differentially expressed (relative up- and down-regulation) in the experimental group. Proteins Fn1, P4ha1, P4ha2, Acan, F2, Col3a1, Fga, Rps29, Rpl34, and Fgg were the *top ten most-connected proteins, implying that miR-1 may regulate an expression network involving these proteins. Of these ten proteins, three were selected for further validation by RT-qPCR: the transcript of Fn1, known to be associated with OA, exhibited relative upregulation in the experimental group, whereas the transcripts of P4ha1 and Acan exhibited relative downregulation. These proteins may thus represent key miR-1 targets during OA-regulatory mechanisms, and may provide additional insights regarding therapeutic mechanisms of miR-1 in context of OA.

P4HA1, transcriptionally activated by STAT1, promotes esophageal cancer progression.

Esophageal cancer (EC) is one of the most frequent cancers with a higher mortality worldwide. Although prolyl 4-hydroxylase alpha polypeptide I (P4HA1) is involved in various human malignancies, the function of P4HA1 in EC remains unclear. The mRNA and protein expressions were assessed by quantitative real-time polymerase chain reaction, western blot and immunohistochemistry. CCK8 assay was used to detect EC cell viability. Cell proliferation was analyzed by colony formation and ethynyl-2'-deoxyuridine assays. In addition, flow cytometry and TdT-mediated dUTP nick-end labeling staining were performed to detect cell apoptosis. Masson's trichrome staining was used to assess the collagen fiber level in tumor tissues. The interaction between STAT1 and P4HA4 was analyzed using ChIP, dual-luciferase reporter gene and Y1H assays. P4HA1 was overexpressed in EC, and its knockdown suppressed EC cell proliferation and collagen synthesis and increased cell apoptosis. Meanwhile, P4HA1 knockdown could repress EC tumor growth in vivo. Our further research displayed that STAT1 promoted P4HA1 expression by interacting with P4HA1 promoter. As expected, P4HA1 overexpression abolished STAT1 knockdown's repression on EC cell malignant behaviors. Our research proved that P4HA1 was transcriptionally activated by STAT1, thereby promoting EC progression.

High P4HA1 expression is an independent prognostic factor for poor overall survival and recurrent-free survival in head and neck squamous cell carcinoma.

BACKGROUND: Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) plays a critical role in modulating the extracellular matrix and promoting tumor progression in various cancers. However, the association between P4HA1 and head and neck squamous cell carcinomas (HNSCC) has not been thoroughly elucidated to date. METHODS: P4HA1 mRNA and protein expression in cancer and normal tissues were analyzed using The Cancer Genome Atlas (TCGA), Gene Expression Omnibus, and Human Protein Atlas databases. Quantitative PCR was applied to determine P4HA1 mRNA expression levels in 162 paired HNSCC and adjacent normal tissues. The cBioPortal for Cancer Genomics was utilized to explore P4HA1 genetic alterations in HNSCC. Then, KEGG analysis of P4HA1 co-expressed genes in HNSCC was conducted using ClueGo in Cytoscape. RESULTS: P4HA1 mRNA and protein levels were significantly increased in HNSCC tissues compared with normal tissues. High P4HA1 expression in HNSCC tissues was significantly associated with tumor category, lymphatic metastasis and pathological stage. The area under summary receiver operating characteristic curve of TCGA and validation cohort was 0.887 and 0.883, respectively. Moreover, elevated P4HA1 expression was associated with unfavorable OS (HR: 1.728, P = .001) and RFS (HR: 2.025, P = .002) in HNSCC patients. CONCLUSIONS: This integrated analysis provides strong evidence that increasing P4HA1 expression is significantly associated with the carcinogenesis of HNSCC. Additionally, high P4HA1 expression serves as both diagnostic biomarker and independent prognostic factor for poor OS and RFS in HNSCC patients.

P4HA1/HIF1α feedback loop drives the glycolytic and malignant phenotypes of pancreatic cancer.

Hypoxia-inducible factor 1α (HIF1α) activation is profoundly implicated in the initiation and progression of multiple malignant tumors. Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) is the active catalytic component of prolyl 4-hydroxylase and has been reported to promote tumor progression in several cancers. In this study, we revealed that P4HA1 was highly expressed in pancreatic ductal adenocarcinoma (PDAC) and predicted a poor clinical outcome. Notably, elevated expression of P4HA1 in PDAC cells was HIF1α-dependent. Gene set enrichment analysis of The Cancer Genome Atlas (TCGA) cohort demonstrated a close link between P4HA1 expression and glycolysis and hypoxia gene signatures in PDAC. Knockdown of P4HA1 significantly suppressed the glycolytic activity of PDAC cells as revealed by reduced glucose utilization and lactate production. Consistently, there was a close correlation between P4HA1 and glycolysis genes. Moreover, we found that P4HA1 can enhance HIF1α stability, indicating a positive feedback loop between HIF1α and P4HA1 in PDAC. Genetic silencing of P4HA1 significantly inhibited the cell proliferation, chemoresistance, and stemness of PDAC cells. Collectively, our findings identify the P4HA1-HIF1α loop as a critical regulator in glycolysis and oncogenic activities of PDAC and provide a potential target for pancreatic cancer treatment.

In vitro chondrogenic potency of surplus chondrocytes from autologous transplantation procedures does not predict short-term clinical outcomes.

BACKGROUND: Autologous chondrocyte implantation (ACI) has been used over the last two decades to treat focal cartilage lesions aiming to delay or prevent the onset of osteoarthritis; however, some patients do not respond adequately to the procedure. A number of biomarkers that can forecast the clinical potency of the cells have been proposed, but evidence for the relationship between in vitro chondrogenic potential and clinical outcomes is missing. In this study, we explored if the ability of cells to make cartilage in vitro correlates with ACI clinical outcomes. Additionally, we evaluated previously proposed chondrogenic biomarkers and searched for new biomarkers in the chondrocyte proteome capable of predicting clinical success or failure after ACI. METHODS: The chondrogenic capacity of chondrocytes derived from 14 different donors was defined based on proteoglycans staining and visual histological grading of tissues generated using the pellet culture system. A Lysholm score of 65 two years post-ACI was used as a cut-off to categorise "success" and "failure" clinical groups. A set of predefined biomarkers were investigated in the chondrogenic and clinical outcomes groups using flow cytometry and qPCR. High-throughput proteomics of cell lysates was used to search for putative biomarkers to predict chondrogenesis and clinical outcomes. RESULTS: Visual histological grading of pellets categorised donors into "high" and "low" chondrogenic groups. Direct comparison between donor-matched in vitro chondrogenic potential and clinical outcomes revealed no significant associations. Comparative analyses of selected biomarkers revealed that expression of CD106 and TGF-ß-receptor-3 was enhanced in the low chondrogenic group, while expression of integrin-α1 and integrin-ß1 was significantly upregulated in the high chondrogenic group. Additionally, increased surface expression of CD166 was observed in the clinical success group, while the gene expression of cartilage oligomeric matrix protein was downregulated. High throughput proteomics revealed no differentially expressed proteins from success and failure clinical groups, whereas seven proteins including prolyl-4-hydroxylase 1 were differentially expressed when comparing chondrogenic groups. CONCLUSION: In our limited material, we found no correlation between in vitro cartilage-forming capacity and clinical outcomes, and argue on the limitations of using the chondrogenic potential of cells or markers for chondrogenesis as predictors of clinical outcomes.

MiR-122 inhibits epithelial mesenchymal transition by regulating P4HA1 in ovarian cancer cells.

Ovarian cancer is one of the most common gyneacologic malignancies, with high morbidity and high mortality. Hsa-miR-122-5p (miR-122) has been reported with tumor-suppressing roles in various cancers. In this study, miR-122 was overexpressed in ovarian cancer cells, and phenotypic experiments demonstrated that miR-122 inhibited migration and invasion in SKOV3 and OVCAR3 cells. MiR-122 also suppressed epithelial mesenchymal transition (EMT), evidenced by expression changes of E-cadherin, vimentin, matrix metalloproteinase (MMP)2, and MMP14. Prolyl-4-hydroxylase subunit alpha-1 (P4HA1) was identified as a target of miR-122, and downregulated by miR-122. MiR-122-induced the elevation of migration, invasion, and EMT were recovered by P4HA1. Additionally, miR-122 restrained the tumor metastasis of SKOV3 cells in peritoneal cavity of nude mice. In summary, we demonstrated that miR-122 inhibited migration, invasion, EMT, and metastasis in peritoneal cavity of ovarian cancer cells by targeting P4HA1 for the first time, which shed lights on the discovery of miR-122 and P4HA1 as possible potential diagnostic markers and therapeutic targets for ovarian cancer.

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA.

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3'-untranslated region (3'UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3'UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis.

Multilevel chitosan-gelatin particles loaded with P4HA1 siRNA suppress glioma development.

It has been reported that prolyl 4-hydroxylase subunit alpha 1 (P4HA1) promoted tumor growth and metastasis of glioma; thus, targeting P4HA1 may be a promising therapeutic strategy against glioma. In consideration of the instability of siRNA in vivo, the chitosan-gelatin microspheres loaded with P4HA1 siRNA (P4HA1 siRNA@CGM) were employed. Firstly, the gel electrophoresis and hemolytic test were performed to assess the stability and blood compatibility of P4HA1 siRNA@CGM. Then, methyl thiazolyl tetrazolium (MTT), cell colony formation, Transwell assay, wound healing assay, gliosphere formation, tube formation, and Western blot were performed to assess the effects of P4HA1 siRNA@CGM on the biological functions of glioma. Finally, 125I-labeled P4HA1 siRNA@CGM was injected into the xenograft mice, radionuclide imaging was recorded, Ki67 and terminal deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) staining was performed to assess the effects of P4HA1 siRNA@CGM on tumor growth and apoptosis of glioma in vivo. The results showed that P4HA1 siRNA and P4HA1 siRNA@CGM not only markedly inhibited the proliferation, metastasis, gliosphere formation, and the protein levels of interstitial markers (N-cadherin and vimentin) and the transcription factors of epithelial-mesenchymal transition (EMT) (Snail, Slug, and Twist1) in glioma cells, but also inhibited the tube formation in human brain microvascular endothelial cells (HBMECs), and P4HA1 siRNA@CGM exhibited the better inhibitory effects than P4HA1 siRNA. Above results suggested the feasibility of P4HA1 siRNA@CGM in the clinical treatment of glioma.

HIF1α/ATF3 partake PGK1 K191/K192 succinylation by modulating P4HA1/succinate signaling in glioblastoma.

BACKGROUND: Hypoxia is a pathological hallmark in most cancers, including glioblastoma (GBM). Hypoxic signaling activation and posttranslational modification (PTM) of oncogenic proteins are well-studied in cancers. Accumulating studies indicate glycolytic enzyme PGK1 plays a crucial role in tumorigenesis, yet the underlying mechanisms remain unknown. METHODS: We first used ChIP assays to uncover the crosstalk between HIF1α and ATF3 and their roles in P4HA1 regulation. Protein degradation analysis, LC-MS/MS, and in vitro succinate production assays were performed to examine the effect of protein succinylation on GBM pathology. Seahorse assay measured the effects of PGK1 succinylation at K191/192 or its mutants on glucose metabolism. We utilized an in vivo intracranial mouse model for biochemical studies to elucidate the impact of ATF3 and P4HA1 on aerobic glycolysis and the tumor immune microenvironment. RESULTS: We demonstrated that HIF1α and ATF3 positively and negatively regulate the transcription of P4HA1, respectively, leading to an increased succinate production and increased activation of HIF1α signaling. P4HA1 expression elevated the succinate concentration, resulting in the enhanced succinylation of PGK1 at the K191 and K192 sites. Inhibition of proteasomal degradation of PGK1 by succinylation significantly increased aerobic glycolysis to generate lactate. Furthermore, ATF3 overexpression and P4HA1 knockdown reduced succinate and lactate levels in GBM cells, inhibiting immune responses and tumor growth. CONCLUSION: Together, our study demonstrates that HIF1α/ATF3 participated in P4HA1/succinate signaling, which is the major regulator of succinate biosynthesis and PGK1 succinylation at K191 and K192 sites in GBM. The P4HA1/succinate pathway might be a novel and promising target for aerobic glycolysis in GBM.

Collagen IV assembly is influenced by fluid flow in kidney cell-derived matrices.

Kidney podocytes and endothelial cells assemble a complex and dynamic basement membrane that is essential for kidney filtration. Whilst many components of this specialised matrix are known, the influence of fluid flow on its assembly and organisation remains poorly understood. Using the coculture of podocytes and glomerular endothelial cells in a low-shear stress, high-flow bioreactor, we investigated the effect of laminar fluid flow on the composition and assembly of cell-derived matrix. With immunofluorescence and matrix image analysis we found flow-mediated remodelling of collagen IV. Using proteomic analysis of the cell-derived matrix we identified changes in both abundance and composition of matrix proteins under flow, including the collagen-modifying enzyme, prolyl 4-hydroxylase (P4HA1). To track collagen IV assembly, we used CRISPR-Cas9 to knock in the luminescent marker HiBiT to the endogenous COL4A2 gene in podocytes. With this system, we found that collagen IV was secreted and accumulated consistently under both static and flow conditions. However knockdown of P4HA1 in podocytes led to a reduction in the secretion of collagen IV and this was more pronounced under flow. Together, this work demonstrates the effect of fluid flow on the composition, modification, and organisation of kidney cell-derived matrix and provides an in vitro system for investigating flow-induced matrix alteration in the context of kidney development and disease.

Comprehensive analysis revealed P4Hs as new biomarkers for prognosis and immunotherapy in head and neck cancer.

Prolyl 4-hydroxylases (P4Hs) are a family of key modifying enzymes in collagen synthesis. P4Hs have been confirmed to be closely associated with tumor occurrence and development. However, the expression of P4Hs in head and neck cancer (HNSC) as well as its relationship with prognosis and tumor immunity infiltration has not yet been analyzed. We investigated the transcriptional expression, survival data, and immune infiltration of P4Hs in patients with HNSC from multiple databases. P4H1-3 expression was significantly higher in HNSC tumor tissues than in normal tissues. Moreover, P4HA1 and P4HA2 were associated with tumor stage, patient prognosis, and immune cell infiltration. P4HA3 was related to patient prognosis and immune cell infiltration. Correlation experiments confirmed that P4HA1 may serve as a prognosis biomarker and plays a role in the progression of nasopharyngeal carcinoma. These findings suggest that P4HA1-3 may be a novel biomarker for the prognosis and treatment of HNSC, which is expected to support the development of new therapies for patients with head and neck tumors and improve patient outcomes.

P4HA1 activates HMGCS1 to promote nasopharyngeal carcinoma ferroptosis resistance and progression.

Ferroptosis is a novel type of iron-dependent regulatory cell death. To date, the regulatory mechanism of ferroptosis in nasopharyngeal carcinoma (NPC) remains poorly understood. In this study, we found that the prolyl 4-hydroxylase (P4H) subunit P4HA1 protects NPC cells from erastin-induced ferroptosis by activating HMGCS1, a key enzyme in the mevalonate pathway. We also found that the P4HA1/HMGCS1 axis promoted NPC cell proliferation in vitro. In vivo, downregulation of the P4HA1/HMGCS1 axis inhibited the growth of NPC cell xenografts and enhanced the inhibitory effect of erastin on tumor growth. Extracellular matrix (ECM) detachment is an important trigger for ferroptosis. We found that the P4HA1/HMGCS1 axis promoted the ferroptosis resistance and survival of ECM-detached NPC cells. In vivo, downregulation of the P4HA1/HMGCS1 axis inhibited the lung colonization of NPC cells and enhanced the inhibitory effect of erastin on NPC lung metastasis. Moreover, the high expression of P4HA1 predicted a poor prognosis and served as a potential independent prognostic factor in patients with NPC. In conclusion, P4HA1 is a novel molecular marker of NPC ferroptosis resistance and a poor prognosis, and the P4HA1/HMGCS1 axis provides a new target for the treatment of NPC progression.

Prolyl 4-hydroxylase P4HA1 Mediates the Interplay Between Glucose Metabolism and Stemness in Pancreatic Cancer Cells.

INTRODUCTION: Cancer stem cells (CSCs) are profoundly implicated in tumor initiation and progression as well as drug resistance and tumor recurrence of many cancer types, especially pancreatic ductal adenocarcinoma (PDAC). Previously, we revealed that prolyl 4-hydroxylase subunit alpha 1 (P4HA1) enhances the Warburg effect and tumor growth in PDAC. However, the possible connection between P4HA1 and cancer stemness in PDAC remains obscure. In this study, P4HA1-dependent cancer stemness was studied by sphere-formation assay and detection of stemness markers. METHODS: Glycolytic capacity in cancer stem cells and their parental tumor cells was investigated by glucose uptake, lactate secretion, and expression of glycolytic genes. Glycolysis inhibitors were used to determine the link between cancer stemness and glycolysis. A subcutaneous xenograft model was generated to investigate P4HA1-induced stemness and glycolysis in vivo. RESULTS: We revealed that ectopic expression of P4HA1 increased the stemness of PDAC cells as evidenced by the increased proportion of CD133+ cells, elevated sphere-formation ability, and the upregulated levels of cancer stemness-related proteins (SOX2, OCT4, and NANOG). Blocking tumor glycolysis with 2-Deoxy-D-glucose (2-DG) or a selective inhibitor of glucose transporter 1 (STF-31) significantly reduced the stem properties of PDAC cells, suggesting that P4HA1-induced glycolysis was essential for the stem-like phenotype of PDAC cells. In addition, in vivo study reaffirmed a promotive effect of P4HA1 on tumor glycolysis and cancer stemness. CONCLUSION: Collectively, our findings suggest that P4HA1 not only affects tumor metabolic reprogramming but also facilitates cancer stemness, which might be exploited as a vulnerable target for PDAC treatment.

The Role of P4HA1 in Multiple Cancer Types and its Potential as a Target in Renal Cell Carcinoma.

Background: Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) provides the majority of the catalytic site of the active P4H enzyme. Emerging evidence has revealed that P4HA1 participates in the initiation and development of several malignant tumors. However, a pan-cancer analysis of P4HA1 has not been performed. Methods: In this study, we carried out an in-depth analysis of the expression patterns and prognostic value of P4HA1 using the datasets of The Cancer Genome Atlas (TCGA) and Kaplan-Meier Plotter. Genomic and epigenetic alterations of P4HA1 and the correlation of P4HA1 with DNA methylation in different cancers were also analyzed across multiple databases. In addition, the purity-adjusted partial Spearman's correlation test was utilized to evaluate the correlation between P4HA1 expression and immune cell infiltration. We also further explored the biological function and mechanism of P4HA1 in renal cell carcinoma (RCC). Results: We characterized the expression profiles and prognostic values of P4HA1 in multiple cancer types. P4HA1 expression was increased in clear cell renal cell carcinoma (RCC) compared to adjacent normal tissues, and P4HA1 positively correlated with the overall survival (OS) and disease-free survival (DFS) in papillary RCC. In addition, a positive correlation between P4HA1 expression and immune cell infiltration was observed in clear cell RCC. We also identified a strong correlation between P4HA1 expression and immune checkpoint gene expression, microsatellite instability, and tumor mutation burden in chromophobe RCC. Finally, the results of in vitro experiments verified that overexpression of P4HA1 promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition of RCC cells. Conclusion: Overall, our study has suggested that P4HA1 might play a significant role in tumorigenesis in RCC and may be a prognostic biomarker and therapeutic target for several malignant tumors, including RCC.

P4HA1 Regulates CD31 via COL6A1 in the Transition of Glioblastoma Stem-Like Cells to Tumor Endothelioid Cells.

Glioblastoma multiforme (GBM) is a common intracranial malignancy characterized by abundant and aberrant vasculature. The efficiency of existing antivascular treatments remains unsatisfactory. The transition of glioblastoma stem-like cells (GSCs) into tumor endothelioid cells (ECs) has been thought to cause glioma neovascularization and anti-angiogenesis tolerance, but the mechanisms regulating glioma transdifferentiation remains unclear. Our previous study found that P4HA1 regulates GSCs vascular mimicry in a hypoxic microenvironment, but the detailed molecular mechanism has not been determined. In this study, candidate protein COL6A1 was screened by mass spectrometry. In vitro experiments show that P4HA1 regulates the expression of CD31 via COL6A1, with the levels of expression of P4HA1, COL6A1 and the vascular endothelial molecular markers CD31 showing positive correlations in vivo assay. Altering the expression of P4HA1 in GSCs altered the expression of COL6A1 and CD31, thereby inducing glioma angiogenesis. In conclusion, this study revealed that the P4HA1/COL6A1 axis modulates the transdifferentiation process of GSCs into ECs. Interrupting this signaling axis can inhibit glioma angiogenesis, suggesting that this axis may be a novel target for antivascular therapy in patients with glioma.

Identification of a novel glycolysis-related gene signature for predicting the prognosis of osteosarcoma patients.

Glycolysis ensures energy supply to cancer cells, thereby facilitating tumor progression. Here, we identified glycolysis-related genes that could predict the prognosis of patients with osteosarcoma. We examined 198 glycolysis-related genes that showed differential expression in metastatic and non-metastatic osteosarcoma samples in the TARGET database, and identified three genes (P4HA1, ABCB6, and STC2) for the establishment of a risk signature. Based on the signature, patients in the high-risk group had poor outcomes. An independent Gene Expression Omnibus database GSE21257 was selected as the validation cohort. Receiver operating characteristic curve analysis was performed and the accuracy of predicting the 1- and 3-year survival rates was shown by the areas under the curve. The results were 0.884 and 0.790 in the TARGET database, and 0.740 and 0.759 in the GSE21257, respectively. Furthermore, we applied ESTIMATE algorithm and performed single sample gene set enrichment analysis to compare tumor immunity between high- and low-risk groups. We found that the low-risk group had higher immune scores and immune infiltration levels than the high-risk group. Finally, we chose P4HA1 as a representative gene to verify the function of risk genes in vitro and in vivo and found that P4HA1 could promote the metastasis of osteosarcoma cells. Our study established a novel glycolysis-related risk signature that could predict the prognosis of patients with osteosarcoma.

P4HA1 Down-Regulation Inhibits Glioma Invasiveness by Promoting M1 Microglia Polarization.

BACKGROUND: Polarization of microglia cells in the glioma microenvironment is closely related to the malignant progression and invasion of gliomas. Prolyl 4-hydroxylase subunit α1 (P4HA1) is the rate-limiting subunit of prolyl 4-hydroxylase (P4H). In previous studies, we showed that P4HA1 could promote the proliferation, migration, and invasion of glioma cells, but the specific mechanisms through which this occurs have not been fully elucidated. MATERIALS AND METHODS: Interactions between glioma and microglia cells were analyzed using bioinformatics. Then, co-culture models were used to obtain conditioned media. To characterize microglial cell polarization, we used PCR and immunofluorescence. Proliferation and invasion assays were used to explore the biological behavior of glioma cells affected by microglia. Finally, marker expression was detected using immunohistochemistry in glioblastoma multiform (GBM) specimens. RESULTS: Knockdown of P4HA1 resulted in reduced chemotaxis of microglia toward GBM cells and increased polarization of microglia toward the M1 phenotype. The changed microglial polarization state, in turn, inhibited the proliferation and invasion of GBM cells. Moreover, in GBM tissue specimens, the P4HA1 expression level is negatively correlated with that of the CD86 microglia M1-specific marker. CONCLUSION: Our results show that P4HA1 promotes immunosuppressive microenvironment formation by cross-talk between GBM and microglia cells and indirectly increases the aggressiveness of GBM.

Comprehensive Analysis of Gene Expression Profiles Identifies a P4HA1-Related Gene Panel as a Prognostic Model in Colorectal Cancer Patients.

Objective: Colorectal cancer (CRC) is the leading cause of mortality worldwide. Growing evidence suggests that the current pathological staging system is inadequate for efficient and accurate prognosis. In this study, we aim to build a prognosis model to predict the survival outcome of CRC patients by using gene expression profiles from The Cancer Genome Atlas (TCGA). Materials and Methods: Univariate and multivariate Cox regression analysis were used to assess the relationship between clinical factors and P4HA1 expression regarding the prognosis of patients with colon adenocarcinoma (COAD). The least absolute shrinkage and selection operator (LASSO) Cox regression model was used to select prognostic differential expression genes (DEGs) for the construction of prognostic risk score model. Kaplan-Meier and receiver operating characteristic (ROC) survival analysis were used to assess the performance of the model on both TCGA cohort and an independent dataset GSE39582. Results: Overexpression of P4HA1 was confirmed to be associated with poor clinical outcome of colon cancer patients in both TCGA and GSE39582 cohorts. Using the TCGA cohort, we identified 1528 DEGs related to elevated P4HA1 expression, and we established a 11-gene panel to construct the prognostic risk score model by LASSO Cox regression analysis based on their expression profiles. The 11-gene signature was further validated in the independent dataset GSE39582. Time-dependent ROC curves indicated good performance of our model in predicting 1, 2, and 3-years overall survival in COAD patients. Additionally, gene set enrichment analysis indicated that the 11-gene signature was related to pathways involved in tumor progression. Conclusions: Together, we have established a 11-gene signature significantly associated with prognosis in COAD patients, which could serve as a promising tool for clinical application in the future.

P4HA1 as an unfavorable prognostic marker promotes cell migration and invasion of glioblastoma via inducing EMT process under hypoxia microenvironment.

This study aims to explore the mechanism of glioblastoma multiforme (GBM) in hypoxia through metabolomic and proteomic analysis. We showed that the migration and invasiveness of LN18 cells was significantly enhanced after 24 h of hypoxia treatment. The metabolomic and proteomic profiling were conducted in LN18 cells cultured under hypoxia condition. Correlation analysis between significant differential metabolites and proteins revealed seven proteins and ten metabolites, of which metabolite L-Arg was negatively correlated with P4HA1 protein. Meanwhile, the expression of HIF1α, nNOS and P4HA1 was up-regulated, and the concentration of L-Arg and NO was decreased and increased respectively. Knockdown of HIF1α reduced the expression of nNOS and P4HA1, the concentration of NO and the invasiveness of cells, while increased the concentration of L-Arg. Similar changes on P4HA1 expression, the concentration of L-Arg and NO were observed when the expression of nNOS was disrupted. Lastly, knockdown of P4HA1 impaired the invasion of LN18 and T98G cells, probably through regulating the expression of Vimentin, MMP2, MMP9, Snail and E-cadherin. Consistent trends on both the overexpression of these relevant genes, as well as the concentration of L-Arg and NO were also observed in all our overexpression experiments. Besides, we investigated the relationship between P4HA1 expression and prognosis by MTA, CGGA and TCGA databases. Increased P4HA1 level was correlated poor prognosis with advanced histological grade. In summary, we found that hypoxia promotes the migration and invasion of GBM via the L-Arg/P4HA1 axis which maybe an effective molecular marker or predictor of clinical outcome in GBM patients.